U.S. patent number 5,063,209 [Application Number 07/565,174] was granted by the patent office on 1991-11-05 for modulation of aids virus-related events by double-stranded rnas.
This patent grant is currently assigned to HEM Research, Inc.. Invention is credited to William A. Carter.
United States Patent |
5,063,209 |
Carter |
* November 5, 1991 |
Modulation of aids virus-related events by double-stranded RNAs
Abstract
The use of mismatched dsRNA, e.g., AMPLIGEN.RTM. for the
manufacture of compositions for use in the selective activation of
a latent natural defense system within human cells, both cells
already infected with AIDS virus as well as cells at risk to
infection. Specific treatments for various clinical phase of the
biological continuum of AIDS virus-related events ranging from
subtle, early immunological lesions to advanced disease are
described. Prophylaxis or prevention of AIDS virus related events,
such as by introduction of mismatched double-stranded RNA into
various blood products or biological fluids to be used in man
(e.g., blood transfusion) or around man (e.g., dialysis programs)
are also described.
Inventors: |
Carter; William A.
(Birchrunville, PA) |
Assignee: |
HEM Research, Inc. (Rockville,
MD)
|
[*] Notice: |
The portion of the term of this patent
subsequent to January 2, 2006 has been disclaimed. |
Family
ID: |
27503824 |
Appl.
No.: |
07/565,174 |
Filed: |
August 10, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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433445 |
Nov 13, 1989 |
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124572 |
Nov 24, 1987 |
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900614 |
Aug 26, 1986 |
4795744 |
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886363 |
Jul 17, 1986 |
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769494 |
Aug 25, 1985 |
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Current U.S.
Class: |
514/44R; 514/885;
514/889; 514/908; 514/934 |
Current CPC
Class: |
A61K
31/70 (20130101); A61K 38/21 (20130101); A61K
38/20 (20130101); A61L 2/0082 (20130101); A61K
38/20 (20130101); A61K 2300/00 (20130101); A61K
38/21 (20130101); A61K 2300/00 (20130101); Y10S
514/934 (20130101); Y10S 514/908 (20130101); Y10S
514/889 (20130101); Y10S 514/885 (20130101) |
Current International
Class: |
A61K
38/21 (20060101); A61K 38/20 (20060101); A61L
2/00 (20060101); A61K 031/70 (); A61K 037/00 () |
Field of
Search: |
;514/44,885,889,908,934 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
May 1984 Press Release. .
Ampligen Research Program fact sheet, May 1984. .
SCRIP Work Pharmaceutical News, Jul. 30, 1984. .
SCRIP World Pharmaceutical News, Jun. 3, 1985. .
Hahnemann University, "ALUMNI" magazine, fall 1984. .
"The Pink Sheet", vol. 46, No. 21, pp. T&G-3,4, May 21, 1984.
.
UPI report of May 16, 1984. .
UPI wire service news reports under various by-lines, May 15 and
16, 1984. .
Longo et al., Ann. NY Acd. Sci., 437:421-30. .
Spiegel, Serminars in Oncology, 14:1, 1987. .
Krigel et al., 1985, Journal of Biological Response Modifiers,
4:358-364. .
Friedland et al., monograph TH.4.6 of Jun. 4, 1987, 3rd
International Conference on Acquired Immunodeficiency Syndrome
(AIDS). .
Travers (1984) Nature 311: 410. Zameonik et al., (1978) PNAS 75:
280-4. .
Izant et al., (1984) Cell, vol. 36: 1007-16. .
Stephanson et al., (1978) PNAS 75: 285-8..
|
Primary Examiner: Morris; Theodore
Assistant Examiner: Brunsman; David M.
Attorney, Agent or Firm: Nixon & Vanderhye
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation of application Ser. No. 07/433,445, filed
Nov. 13, 1989, now abandoned, which is a continuation of
application Ser. No. 07/124,572, filed Nov. 24, 1987, now
abandoned, which is a continuation-in-part of application Ser. No.
900,614 filed Aug. 26, 1986 now U.S. Pat. No. 4,795,744, which in
turn, is a continuation-in-part of application Ser. No. 886,363
filed July 17, 1986 now abandoned, which in turn, is a
continuation-in-part of application Ser. No. 769,494 filed Aug. 25,
1985, now abandoned.
Claims
What is claimed:
1. A method of treating retrovirus or T-cell lymphotropic
virus-induced conditions including AIDS in a person infected with
same comprising administering to that person an amount of dsRNA
sufficient to therapeutically benefit AIDS-related disorders.
2. The method of claim 1 in which the dsRNA is mismatched
dsRNA.
3. The method of claim 2 in which the dsRNA is a complex of a
polyinosinate and a polycytidylate containing of from 1 in 5 to 1
in 30 uracil or guandidine bases.
4. The method of claim 3 in which the mismatched dsRNA is rI.sub.n
.multidot.(C.sub.12 U).sub.n.
5. The method of claim 1 in which the dsRNA is administered in an
amount sufficient to result in a level of 0.01 to 1000 microgram
per milliliter of the patient's body fluid.
6. The method of claim 1 in which an AIDS inhibitor is administered
concurrently with the dsRNA.
7. The method of claim 1 in which a second AIDS virus inhibitor is
concurrently administered to the patient together with the
dsRNA.
8. The method of claim 1 in which a lymphokine is administered with
the dsRNA as an AIDS inhibition assisting agent.
9. A method of treating AIDS in a person infected with same
comprising administering to that person, in combination, an amount
of mismatched dsRNA sufficient to benefit AIDS-related disorders
and an interferon or an interleukin in an amount sufficient to
assist in inhibiting AIDS.
10. The method of claim 9 in which the mismatched dsRNA and the
interferon are administered in an amount sufficient to result in a
ratio of 0.01 to 1000 microgram per milliliter of dsRNA to 0.1 to
100,000 IRU of interferon in the patient's body fluid.
11. A therapeutic composition for the prevention and treatment of
retrovirus or T-cell lymphotropic virus including AIDS in a person
comprising, in combination, a viral-inhibiting amount of dsRNA in
combination with an AIDS-inhibiting amount of interferon, the dsRNA
present in an amount when administered to a person to result in a
level of 0.01 to 1000 micrograms per milliliter of body fluid and
the interferon present in an amount when administered to a person
to result in a level of 0.1 to 100,000 IRU per milliliter of body
fluid.
12. The therapeutic composition of claim 11, in which the dsRNA is
mismatched dsRNA.
13. The therapeutic composition of claim 11 in which the mismatched
dsRNA is rI.sub.n .multidot.(C.sub.12 U).sub.n.
14. A method of rendering a human-originating biological fluid or
cells resistant to infection with retrovirus or T-cell lymphotripic
virus including AIDS virus, enhancing the resistance thereof, or
mitigating the effect of infection therefrom comprising admixing or
contacting said biological fluid or cells with a virus-inhibiting
amount of dsRNA.
15. The method of claim 14 in which the dsRNA is mismatched
dsRNA.
16. The method of claim 14 in which the mismatched dsRNA is
rI.sub.n .multidot.(C.sub.12 U).sub.n.
17. The method of claim 13 in which the biological fluid is human
blood or a fraction thereof, or the cells are NK cells, T-cells,
T-suppressor cells, T-helper cells, spermatozoa, ova or fetal
cells.
18. As a composition of matter, human blood or a fraction thereof
or human cells and an effective amount of a mismatched dsRNA.
19. A process of removing or inactivating retroviruses or T-cell
lymphotropic viruses including AIDS viruses from a device for
handling parentheral fluids or mitigating the effect of infection
therefrom comprising contracting said device with a composition
containing an AIDS-inhibiting amount of a dsRNA.
20. A method of restoring an anergic immune state to a
substantially normal immune state, as measured by skin testing,
said method comprising administering to a person having an anergic
immune state an effective amount of dsRNA.
Description
AIDS (Acquired Immunodeficiency Disease Syndrome) represents a
major public health threat of the Twentieth Century. Because of its
biologically variable nature, its subtle means of spread from
individual to individual, and its "latent" disease period which
defies even the ready detection of its very presence, AIDS has
rapidly evolved into an epidemiologic dilemma of unprecedented
proportions. Indeed, even with the considerable resources already
deployed to increase diagnostic capabilities, the available
technologies still fall well short of the necessary requirements to
gain firm control of this problem within any section of the
population (e.g., see Proceedings of Workshop entitled "Experience
with HTLV-III Antibody Testing, an Update on Screening, Laboratory
and Epidemiological Correlations", sponsored by the Center for
Drugs and Biologics, FDA, National Institutes of Health, and
Centers for Disease Control, held on July 31, 1985, at the National
Institute of Health, Bethesda, Md.; see also, Budiansky, S. in
Nature, volume 316, page 96, July 11, 1985). Two different
designations for the AIDS virus exist; LAV is the designator for
the AIDS virus isolated at the Pasteur Institute, Paris, France and
HTLV-III is the designator for the AIDS virus isolated at the
National Institute of Health, Bethesda, Md., U.S.A. Frequently in
this text, the AIDS virus will be referred to generically or
designated HTLV III or LAV without intending to differentiate
between them. Furthermore, the term "AIDS virus" in the
specification includes any and all other viruses which may be
associated with producing AIDS, whether yet isolated or not.
Current therapeutic considerations have taken one of two
approaches: immunological (vaccine production) or direct attacks on
the virus itself (antiviral therapy). While vaccine production
initially appeared extremely promising, at least in theory, new
knowledge of the viral composition has significantly undermined
this promise; namely, the virus apparently readily mutates or
changes its basic biochemical structure such that critical viral
antigenic "determinants"--necessary for vaccines to be
effective--readily undergo mutation or change. For example,
HTLV-III isolates in California, Maryland and England have recently
been found to be significantly different from each other when their
genomic content(s) has been rigorously analyzed. The implication of
such studies is that vaccination against AIDS will not have
widespread lasting benefits such as the conclusively
beneficial/durable results historically characteristic of most
other vaccines, such as those directed against polio virus, measles
virus, etc.
In the second, or direct antiviral approach, other scientists have
tested several compounds including HPA-23, suramin, ribavirin,
interferon and fascarnet, etc. These compounds have been introduced
into either the laboratory or clinical studies with little-to-no
evidence of therapeutic success to date. Indeed, consistent
evidence of high toxicity and/or severe side-effects has been
reported in nearly every instance (e.g., see summary prepared by M.
Clark, Newsweek, page 71, Aug. 5, 1985; also, C. Wallis, Time,
pages 40-47, Aug. 12, 1985). None of these drugs are indeed new or
able to be selectively directed against the underlying disorder;
namely, multiplication of AIDS virus in certain cells. For example,
HPA-23 is a combination of heavy metals (reminiscent of the use of
arsenic to treat venereal diseases in the 1920s, or pre-penicillin
era); suramin is actually an anti-parasitic (sleeping sickness)
compound and fascarnet is an anti-herpes virus compound. The latter
two compounds may inhibit an AIDS-virus related enzyme termed
"reverse transcriptase", but there is no evidence that such an
enzymatic inhibition, in fact, would result in any therapeutic
improvement or disease prevention/amelioration. Similarly,
interferon, which is as toxic and non-specific as the
above-mentioned compounds, may have some limited activity on
AIDS-virus related tumors, but numerous studies indicate that it
holds little promise as an effective antiviral against AIDS virus,
or most other viruses for that matter.
Mismatched double-stranded RNA is a known form of macromolecular
RNA (see U.S. Pat. Nos. 4,024,222 and 4,130,641) in which
destabilization of the double helix prevents base pairing.
Mismatched dsRNA is well known for its interferon-induction
properties which indicate a mechanism unrelated to interferon
induction per se (e.g., see European Patent Application 83305426.5
filed Aug. 15, 1983, entitled "Antiproliferative Action of dsRNAs
on Tumor Cells"). A typical therapeutic embodiment of mismatched
double-stranded RNA is the synthetic dsRNA formed by complexes of
polyriboinosinic and polyribocytidylic/uridylic acid, such as
rI.sub.n r(C.sub.x,U or G).sub.n where x has a value from 4 to 29,
e.g. rIn.r(C.sub.12 U).sub.n herein referred to as "Ampligen", a
trademark of HEM Research, Inc., of Rockville, Md., U.S.A. Many
mismatched dsRNA polymers which behave similarly to Ampligen have
been studied. The key therapeutic advantage of mismatched dsRNAs
over other forms of natural and/or synthetic dsRNAs is their
reduced toxicity in animals and man. For example, Lampson et al in
U.S. Pat. No. 3,666,646 described earlier complexes of dsRNA which
are capable of triggering various interferon-related effects, but
the toxicity of such compounds precluded any clinical utility in
the treatment of cancer or related disorders.
The inventor has fully described the known activities of various
interferons, interferon inducers and dsRNAs in his recent textbooks
on the subject (e.g., see the inventor's chapter in Anticancer and
Interferon Agents, pages 301 through 319, edited by R. M.
Ottenbrite and G. B. Butler, Marcell Dekker, New York, 1984; also
see Handbook of Experimental Pharmacology on Interferons, edited by
P. E. Come and W. A. Carter, 1984, published by Springer-Verlag,
New York and Heidelberg; pages 535-555 fully describe the known
properties of dsRNAs).
Mismatched dsRNA, e.g., Ampligen, is now known to have therapeutic
activity against certain human tumors, particularly kidney cancer.
Although currently thought of as purely an "interferon inducer",
dsRNAs are active on certain human tumors which are completely
resistant to interferon itself as fully described in European
Patent Application 83305426.5 of which the present applicant is an
inventor.
Similar to the lack of activity against certain tumors, it has now
been demonstrated that virtually all types of interferon exhibit no
significant activity in the treatment of the AIDS virus. Being
known "interferon inducers", it is particularly surprising that
mismatched dsRNAs have application in the treatment of AIDS-related
disorders.
The present invention is based on another new and unexpected
property of dsRNA, especially mismatched dsRNA, as exemplified by
Ampligen, which has a surprisingly wide range of applicability to
the treatment of AIDS-related disorders.
The present invention provides the use of a dsRNA in the
manufacture of a composition for use in the treatment of AIDS by
therapy or prophylaxis.
This invention includes a method of treating retrovirus or T-cell
lymphotropic virus-induced conditions, including AIDS, in a person
infected with same, by administering to that person an amount of
dsRNA sufficient to therapeutically benefit AIDS-related disorders.
A second virus inhibitor, such as interferon, may be concurrently
administered to the patient.
An important symptom of AIDS is the suppression of the normal
immune state as indicated by the immune skin response. This is
restored during successful treatment with dsRNA.
Accordingly, the invention includes the use of dsRNA in the
manufacture of a medicament for restoring an anergic immune state
to a substantially normal immune state as measured by testing the
immune skin response.
The dsRNA may be a mismatched dsRNA.
HTLV III/LAV infection produces a particularly wide range of
severity of response from relatively asymptomatic carriers to
profound immunological paralysis. To treat this range of response,
a wide range of dosage of dsRNA may be appropriate. When low
dosages are employed, the use of non-mismatched dsRNA may be
appropriate and dosages may be sufficiently low to avoid the
adverse reactions mentioned above. Where higher doses are
appropriate the need to use mismatched dsRNA will be greater.
Preferably, the dsRNA is such as to produce an elevated
intracellular concentration of 2'-5'A oligomers without host
toxicity, e.g. due to an accelerated bioavailability, a structure
adapted to activate 2'-5'A oligomer production without producing
side effects, or due to a relatively short half life following
administration.
By "matched dsRNA" are meant those in which hydrogen bonding (base
stacking) between the counterpart strands is relatively intact,
i.e. is interrupted on average less than one base pair in every 29
consecutive base residues. The term "mis-matched dsRNA" should be
understood accordingly.
The dsRNA may be a complex of a polyinosinate and a polycytidylate
containing a proportion of uracil bases or guanidine bases, e.g.
from 1 in 5 to 1 in 30 such bases (poly I. poly (C4-29.times.U or
G).
The dsRNA may be of the general formula rI.sub.n.C(C.sub.12
U).sub.n. Other suitable examples of dsRNA are discussed below.
The composition may comprise the dsRNA and an AIDS inhibition
assisting agent, such as a lymphokine, e.g. an interferon or an
interleukin such as IL-2.
The invention accordingly includes the use of dsRNA in the
manufacture of a pharmaceutical composition for the prevention or
treatment of AIDS in a person comprising, in combination, a dsRNA
and a lymphokine such as an interferon.
Preferably the composition contains the dsRNA and the interferon in
the ratio of 0.01 to 1000 micrograms of dsRNA to 0.1 to 100,000 IRU
of interferon.
The invention further includes a method of rendering a, preferably
human-originating, biological fluid or, preferably
human-originating, cells resistant to viral infection, enhancing
the resistance thereof, or mitigating the effects of infection
therefrom comprising admixing or contacting said biological fluid
or cells with an AIDS-inhibiting amount of a dsRNA.
The biological fluid may be human blood or a fraction thereof for
instance for use in transfusion or dialysis.
The invention further includes as a composition of matter, human
blood or a fraction thereof or human cells and an AIDS-inhibiting
amount of a mismatched dsRNA.
Furthermore, the invention includes also a process of removing or
inactivating AIDS viruses from a device for handling parenteral
fluids or mitigating the effect of infection therefrom comprising
contacting said device with a composition containing an
AIDS-inhibiting amount of a dsRNA.
The invention includes also a composition for use in the treatment
of AIDS comprising a dsRNA and a pharmaceutically acceptable
carrier or diluent together with instructions for use in the
treatment of AIDS.
Thus there are disclosed hereinafter by way of example, therapeutic
methods of selectively inhibiting human viruses, specifically the
AIDS virus, without significant toxicity to normal cells; methods
of inhibiting, delaying or preventing a person from becoming
infected with AIDS virus; methods of preventing or treating
AIDS-related disorders in humans; methods of treating human
biological fluids, cells and tissue products to prevent or arrest
infection or contamination with AIDS virus; and methods of
correcting specific lesions associated with AIDS virus, including
loss of interferon receptors on cells within the immune system,
cutailment or reduction of intracellular 2'-5'A synthetase and the
reduction or loss of intracellular dsRNAs in selected cells of the
immune system. Pharmaceutical compositions for use in these methods
are also disclosed.
The use of dsRNA is adapted to correct specific lesions associated
with AIDS virus, including loss of interferon receptors on various
critical cells within the immune system, curtailment/reduction of
intracellular 2'-5'A synthetase in various critical cells within
the immune/bodily defense system, and reduction or loss of
intracellular dsRNA in various critical cells within the
immune/bodily defense system.
The mismatched dsRNAs preferred for use in the present invention
are based on copolynucleotides selected from poly (C.sub.n,U) and
poly (C.sub.n,G) in which n is an integer having a value of from 4
to 29 and are mismatched analogs of complexes of polyriboinosinic
and polyribocytidilic acids, formed by modifying rI.sub.n. rC.sub.n
to incorporate unpaired bases (uracyl or guanine) along the
polyribocytidylate (rC.sub.n) strand. Alternatively, the dsRNA may
be derived from poly (I). poly (C) dsRNA by modifying the ribosyl
backbone of polyriboinosinic acid (rI.sub.n) e.g. by including
2'-O-methyl ribosyl residues. These mismatched analogs of
rI.sub.n.rC.sub.n, preferred ones of which are of the general
formula rI.sub.n. r(C.sub.12,U).sub.n, are described by Carter and
Ts'o in U.S. Pat. Nos. 4,130,641 and 4,024,222 the disclosures of
which are hereby incorporated by reference. The dsRNA's described
therein generally are suitable for use according to the present
invention.
Other examples of mismatched dsRNA for use in the invention
include:
poly (I). poly (C.sub.4, U)
poly (I). poly (C.sub.7, U)
poly (I). poly (C.sub.13, U)
poly (I). poly (C.sub.22, U)
poly (I). poly (C.sub.20, G)
poly (I). poly (C.sub.29, G) and
poly (I). poly (C.sub.p) 23 G>p
Pharmaceutical compositions in accordance with this invention
include the mismatched dsRNA, optionally also an interferon,
together with a pharmaceutically acceptable carrier or diluent.
Pharmaceutical compositions contemplated by this invention include
those adapted for parenteral administration in a suitable
pharmaceutical vehicle.
Thus, for example, parenteral solutions, suspensions and
dispersions can be prepared as required according to known
pharmaceutical techniques with sterile or pyrogen-free water as the
solvent/diluent optionally also with physiologically acceptable
salts.
It will be understood that the absolute quantity of active
ingredients present in any dosage unit should not exceed that
appropriate to the rate and manner of administration to be
employed, but on the other hand should also desirably be adequate
to allow the desired rate of administration to be achieved by a
small number of doses. The rate of administration will moreover
depend on the precise pharmacological action desired.
The amount of mismatched dsRNA administered is preferably
sufficient to achieve a level of from 0.01 micrograms per
millilitre of body fluid after equilibration of the dsRNA level
through the body fluid up to 1000 micrograms per milliliter in the
systemic circulation immediately following administration. When
concurrently administered, interferon is preferably included in an
amount that results in a level of 0.1 to 100,000 IRU per milliliter
of body fluid. Generally, the amount to be administered will depend
on the severity of the condition, in particular the level of
available intracellular bioactive dsRNA, 2'-5' oligo A molecules or
2'-5'A synthetase. As used herein, the term body fluid is the
solution of serum, vitamins, etc. which circulates within the
organism and bathes the tissues. Expected body fluid volumes of
patients are of course known to practitioners and are published as
charts and tables, which are routinely available.
As indicated above, the invention includes the treatment of
biological fluids with dsRNA and similarly the treatment of cells
with dsRNA. Blood products such as whole blood used in transfusions
or components of whole blood, such as "packed (concentrated) red
blood cells, packed white blood cells, platelet concentrates or
serum protein fractions such as immunoglobulins" may be treated in
this way. Such blood products may have mismatched dsRNA at
appropriate concentrations added to them at the time of initial
isolation and prior to any cryopreservation.
Alternatively, an effective concentration may be added to the blood
product immediately prior to injection into the recipient. In such
cases, the operator may simply refer to a standard table of body
fluid volumes which interrelate the weight of the recipient to his
or her body fluid volume, which is the total of the body fluid
volume of the patient and the body fluid volume available for
equilibration with the necessary quantity of the dsRNA. A sixty to
seventy kilogram patient will have a body fluid volume of
approximately five to six liters.
Blood products as described above may have undetected retroviruses,
especially AIDS-related virus associated with them. The objective
is to provide a final concentration in the blood product in
question which prevents the "seeding" of the occult retroviruses in
the diverse tissues of the recipient of the blood product. By
preventing a successful seeding of such retrovirus one can prevent
what otherwise would become a life threatening viral proliferation
when the contaminated transfused cells and/or cell products are
admixed and/or distributed throughout the recipient's system.
Other important uses for this technique are instances where the
patient's blood is transitorily exposed to donor blood and/or blood
products, such as through a membrane which may be defective or may
serve to transmit retroviruses, examples of such instances include
the use of equipment such as extracorporeal pumps and associated
devices used during cardiac surgery, cardiac bypass surgery, organ
transplants and so forth. This differs from the situation in which
whole blood or blood products are infused into the patient.
Transitory exposure through a membrane involves only temporary
exposure to possibly contaminated blood, exposure lasting as little
as a few minutes or for as long as several hours depending on the
length of the surgical or other medical procedure. Similar exposure
is possible with renal dialysis equipment in which the presence of
an effective concentration of dsRNA will thwart the possibility
that undetected retroviruses may traverse membranes and seed the
body of an individual connected to such equipment.
Medical literature also documents the passage of the AIDS-related
virus through nasal and lachrynal secretion. Medical equipment and
devices having the potential to transfer retroviruses from one
patient to another are also effectively treated with mismatched
dsRNAs according to the invention. Illustrative examples include
inhalation and inhalation therapy equipment used to assist in
respiration as well as instruments used to examine and treat
eyes.
Aerosols containing dsRNAs may be sprayed onto the contacted
portions of the device to prevent contamination of equipment due to
multiple users.
The amount of dsRNA included in or added to whole blood or blood
products will depend on the overall dilution rate of the blood or
blood products in use. First, the tissue volume of the patient may
be determined based upon the size of the patient, then the quantity
of dsRNA may be calculated. A transfused blood product will require
a greater concentration of the dsRNA because of the extensive
overall dilution as compared with an extracorporeal blood pump or
dialysis equipment using a membrance in which the donor patient is
to be protected without concern for dilution to immobilise any
retrovirus in the donated material. Preferred amounts for
non-diluted uses are in the range of from about 0.1 microgram to
about 200 micrograms per milliliter of body fluid. In contrast, for
whole blood, usually available in containers or 350 and 500 ml, a
sixty kilogram patient with a whole body fluid volume of between
about five and six liters will require about 200 mg of dsRNA to
achieve a concentration of 40 micrograms per milliliter upon
equilibration.
The invention will be illustrated by the following description of
examples of the use of dsRNA.
Mismatched dsRNAs, e.g., Ampligen, were formulated in aqueous
solution such that, when added to various human cells, transient
concentrations of 0.01 microgram to 250 microgram per milliliter of
fluid (bathing cells) was obtained. A variety of different cells,
all potential targets for infection with HTLV-III (AIDS virus),
were used.
Below described is a typical experiment with H-9 cells, a human
lymphoid cell which can be acutely or chronically infected with
HTLV. The cells were grown under standard conditions (e.g., see
Mitsuya et al, Science volume 226, page 172, 1984) and analyzed for
several weeks with respect to the presence of HTLV enzymes or
HTLV-specific proteins. Various concentrations of Ampligen were
added, before, after or simultaneously with the virus to thus mimic
various clinical conditions. Most importantly, careful analyses of
cell number, morphology, etc., were done to determine if the
mismatched dsRNA had various non specific effects on lymphoid cell
growth as had been described by Mitsuya with other inhibitors.
Cells, at different times during the experiment, were isolated and
studied by HPLC analysis of the 2',5'-A oligomers using methods
described by Lee et al (Biochemistry, volume 24, page 551, 1985).
Certain A oligomers, when present in nature, are known to confer
viral resistance to cells, but it has not been previously
described, or anticipated, that a test compound (synthesized by
man) could precisely trigger this natural reaction selectively, and
thus strengthen a natural defense mechanism against viruses in
general, and AIDS virus in particular.
TABLE 1 ______________________________________ Effect of Ampligen
on HTLV-III Infectivity % HTLV Positive Cells Reverse Transcriptase
Day + Drug - Drug + Drug - Drug
______________________________________ Experiment A 4 0 50 1,412
24,287 9 7 90 144,632 1,243,300 Experiment B 3 0 0 353 323 7 0 0
400 1,200 10 0 5 800 2,261 14 2 >80 1,100 112,247 17 5 >90
1,200 1,560,000 ______________________________________
Experiment A was conducted with 25 times more virus (expressed as
infectious units) than target cells whereas Experiment B was
conducted with 10 times more cells (designated H-9 cells) than
virus. Percent cells positive refers to cells which expressed
HTLV-III markers designated p24 and p19 as determined by
immunofluorescence; reverse transcriptase refers to a viral enzyme
measured in the cell supernatant by the standard template
designated poly rA/dT. Concurrently, cell counts were done: the
cells multiplied at normal growth rates at all concentrations of
Ampligen tested (up to 300 micrograms per milliliter). In
experiments A and B, Ampligen (50 micrograms per milliliter) was
added on day 1. HPLC analysis showed (by day 3-7 in either
Experiment A or B) a specific shift in the 2',5'-oligomer profile
such that the higher (less antiviral) molecular weight (MW)
oligomers shifted to the lower (most antiviral) MW oligomers which
contributed to a selective and strong suppression of the AIDS
virus.
Similar experiments were conducted with various other human cells
which are potential in vivo targets for AIDS virus: These
experiments included other cells functionally determined to be NK
(natural killer) cells, T helper cells, T suppressor cells and
mononuclear cells, etc. In all instances, various concentrations of
mismatched dsRNA were able to selectively arrest and/or prevent
HTLV multiplication without any effect on normal cell growth and
maturation. Proliferation of the T cells in culture was maintained
by the standard addition of IL-2 factor as well known to those
familiar in the art of cell biology. The selective suppression of
HTLV by mismatched dsRNA was consistently associated with a
specific shift, or enhancement, in the 2',5'-oligomer profile as
determined by HPLC analysis.
COMPARISONS OF MISMATCHED dsRNAs WITH INTERFERON
Since dsRNAs, being "interferon inducers", might be viewed as
simply working through (via) an interferon mechanism, any activity
of interferon per se might be thought to suggest activity by dsRNA.
Thus, the following experiments establish that uniqueness of the
present invention by demonstrating no significant activity of
interferons of virtually all types in the face of very potent and
specific anti-AIDS virus activity by Ampligen.
CEM (another human T cell line) cells were treated with either 250
I.U./ml recombinant alpha interferon, 250 I.U./ml natural beta
interferon, 50 I.U./ml natural gamma interferon of 50 .mu.g/ml
Ampligen for 18 hours prior to infection with LAV. After 15 days of
culture, the following data were collected.
TABLE 2 ______________________________________ Effect of Ampligen
on LAV Infectivity Reverse tran- indirect immunofluores- scriptase
[cpm/ml cence [% of cells with culture fluid] LAV antibody binding]
______________________________________ untreated 1,147,000 49%
alpha interferon 503,000 5% beta interferon 1,500,000 60% gamma
interferon 360,000 30% Ampligen 24,000 <1%
______________________________________
The data indicate a profound inhibition of viral replication by
Ampligen.
Thus, dsRNA (Ampligen) inhibits (>99%) HTLV-III/LAV infection in
H9 (T4) cells and CEM (T4) cells. Interferons alpha, beta and gamma
are marginally active or inactive. Viral inhibition by Ampligen is
very selective and occurs without any measurable effect on cell
growth.
Thus, Ampligen inhibits HTLV-III/LAV virus replication by a
mechanism distinct from interferon. This finding suggests that
Ampligen and interferon or other drugs could be used in
combination, whereby Ampligen would inhibit viral replication and
the interferon could be used in stimulating the immune system to
restore pre-disease function.
In the restoration of the natural antiviral state Ampligen acts
distal to the AIDS/ARC block in the antiviral pathway to restore a
natural antiviral RNase activity which destroys the viral
genome.
Therefore, the drug has the potential to inhibit viral replication,
as well as to augment the immune response. Thus, it should be noted
that Ampligen is not simply a reverse transcriptase inhibitor of
the virus, but acts through the dual mechanisms of human immune
system stimulation and establishment of an antiviral state in
target cells. Ampligen is readily set apart from interferons, in
that it may well be able to cure viral infections and viral induced
tumors in animals which are completely refractory to exogenous
interferon therapy. These observations distinguish Ampligen from
many other drugs proposed for use against this disease.
In related studies, the pathophysiology of AIDS was studied and
unexpected and previously undetected biochemical lesions in
individuals predisposed (homosexual) to AIDS were detected. Such
lesions are correctable by Ampligen and explain its potent
antiviral activity in the face of nominal activity (if any), by
interferons or most other compounds which have been tested.
First, it was observed that the missing critical biochemical
co-factor was dsRNA, in the T lymphocytes of each of 6 individuals
predisposed to AIDS (homosexual males) and in each of 6 AIDS
victims. Measurements were made of dsRNAs by standard techniques
following cell disruption. The effect of Ampligen can thus be
accurately viewed as specific replacement therapy for a critical
biochemical entity needed for humans to withstand virus attack at
the subcellular level. When dsRNA is present naturally or added, as
via Ampligen therapy, an active RNase L is formed which then
"eats-up" (hydrolizes), or destroys, the viral mRNA thus stopping
or containing the viral infection. Subsequently to the filing of
the USA priority application, Preeble et al reached a complementary
conclusion (September 1985 issue of Journal of Infectious Diseases,
pages 457-465, entitled "Interferon Induced 2'-5' Oligoadenylate
Synthetase During Interferon Alpha Therapy in Homosexual Men with
Kaposi's Sarcoma: Marked Deficiency in Biochemical Response to
Interferon in Patients with Acquired Immunodeficiency Syndrome").
They showed that the addition of interferon to immune cells from
AIDS victims did not result in the expected boost in this specific
intracellular pathway, a first line of defense against various
viruses.
Second, it was determined that AIDS virus infection itself caused a
further loss, or alteration, in the interferon receptor on the cell
surface such that interferon could not bind well and could not
therefore inititiate the critical "cascade" of biochemical events
normally leading to arrest of viral infection and
restoration/maintenance of immune competence. It was further
observed that unusually low levels of 2'-5'A synthetase follow
HTLV-III infection. Thus, AIDS virus infection itself is
characterized by applicant at least three (3) deleterious events
which however, can be specifically remedied by Ampligen or dsRNA
therapy: (1) loss of interferon receptors, (2) reduction in 2'-5'A
synthetase, and (3) abnormally low intracellular dsRNA.
In the relevant biochemical pathway, dsRNA works downstream or
distal to the biochemical lesions in individuals at risk (with or
without active AIDS virus infection), thus restoring normal
antiviral response capability.
dsRNA is very efficiently taken up by cells and therefore does not
require an intact IFN receptor. Further, mismatched dsRNA is also
available as bioactive fragments to accelerate intracellular uptake
and distribution. Thus, dsRNA has the potential to readily cross
the blood-brain barrier, which may be a residual reservoir of AIDS
virus in some individuals.
Typically, dsRNA for use in the present invention will be of the
molecular size exemplified in U.S. Pat. Nos. 4,024,222 and
4,130,641 but much lower molecular weight dsRNA, obtainable as
fragments of the parent molecule are also effective. These may for
instance be of one half to one tenth the size of the typical dsRNA
materials described previously.
dsRNA activates the 2'-5'A polymerases and promotes the synthesis
of a 500-fold increment in the active antiviral oligonuleotides
over that possible with interferons alone (dsRNA leads to 250
nanomoles of 2'-5' oligo A in 1.6.times.10.sup.8 cells whereas 200
units/ml IFN lead to only 0.5 nanomoles of 2'-5' oligo A.
Similar phenomena were observed by applicant in the NK cells of
individuals at risk to development of AIDS, or possessing frank
AIDS. A similar situation exists with respect to the levels of
Natural Killer cell (NK) activity in AIDS/ARC patients. However,
much less is known regarding the mechanisms of NK regulation.
AIDS/ARC patients and healthy members of "at-risk" groups often
have weak immunosurveillance capacity (functional NK and T
lymphocytes) and cannot be re-activated by interferons. Ampligen
acts distal to the disease block and can activate cytotoxic
lymphocytes.
The above theories have been tested clinically in the following
confidential trial.
A 60 kg adult male with an AIDS-related complex of intermediate
severity (lymph nodes enlarged and unable to eat solid foods for
almost one year due to enlarged lymph nodes, AIDS-virus
concentration greater than 10.sup.5 particles per ml of blood but
having no evidence of tumors or other infections) was treated with
200 mg mismatched dsRNA (Ampligen) admixed with physiologic saline
solution by intravenous drip over a period of 30 minutes. This
infusion resulted in a concentration of approximately 40 mcg (0.04
mg) per ml of body fluid when the dsRNA was completely circulated
throughout the body and fully equilibrated with all of the
extracellular body fluid. The dsRNA was infused into the patient on
6 consecutive treatment intervals spaced 2-3 days apart and was
sufficient to rid the patient's body of all measurable AIDS-related
virus and to correct the patient's deteriorated immune function.
Restoration of the immune function was indicated by a 50%
improvement in the ratio of T4/T8 lymphocytes, a ratio recognized
as a reliable measurement of general immune capability with respect
to retroviruses. Further, at the conclusion of therapy the
patient's immune capability, as measured by skin testing, returned
to normal from the anergic condition that was measured prior to
administration of the mismatched dsRNA. The patient was able to
resume eating solid food at the conclusion of therapy and his
conditions continues to progress.
Therapeutic combinations of mismatched dsRNAs and lymphokines
exhibit enhanced effectiveness in inhibiting viral (HIV)
replication.
Table 3 shows the synergistic activity of dsRNA and lymphokines on
viral replication. A retrovirus (HIV) was exposed to graded doses
of recombinant beta interferon without or with dsRNA. Normally, the
viral infection caused extensive cytolysis but dsRNA at very low
doses (12.5 micrograms/ml) causes substantial residual protection
even after 5 days of viral replication and also had a pronounced
and unexpected enhancing effect on the activity of interferon.
Similar experiments summarized in Table 4 demonstrate the ubiquity
of the effect both with respect to other immune system components
(cells) and other lymphokines.
Lymphokines of particular interest include the interferons, as
discussed above, both naturally occurring and made recombinant
technology. Also the interleukins, specifically interleukin-2
(IL-2) and recombinant interleukin-2 (rIL-2), and tumor necrosis
factor (TNF) may be considered. Also included are lymphokine
activated killer cells (LAK) formed in animals in response to
exposure to a lymphokine.
When interferon (alpha) is used as the lymphokine, an amount of
from 0,01 to 100,000 IRU per milliliter of the patient's body fluid
is provided. When IL-2, preferably rIL-2, is the lymphokine, the
amount administered lies within a range of about 10.sup.2 IL-2
units per kg of the patient's body weight up to a value approaching
unacceptable levels of toxicity in that patient, which may be as
high as 10.sup.6 IL-2 units. However, most effective,
toxic-reaction manageable values are in the range of from about
10.sup.3 to about 10.sup.4 IL-2 per kg of body weight.
TABLE 3 ______________________________________ Anti-HIV Activity of
Combined rIFN-.beta. and Ampligen Treatment in T-Cell Cultures
Percent Protection* rIFN-.beta. 4 Day 5 Day (Units/ml) -Ampl
+Ampl.sup..+-. -Ampl +Ampl ______________________________________
1250 58 100 19 64 625 42 100 8 55 312 31 94 4 44 156 31 90 2 44 78
8 57 2 25 39 5 46 0 17 19.5 2 24 0 11 9.7 0 22 0 5
______________________________________ *Average values, n = 3.
Standard deviation were always within 10% of values. Cells were
incubated in the presence or absence of rIFN.beta. (Triton
Biosciences, Inc.) and Ampligen for 4 hours prior to virus
challenge. Effectors were assayed in 96well microtiter plates using
MT2 as target cells and HTLVIIIB as virus at an M.O.I. of 0.1-1.0.
.sup..+-.Ampligen = 12.5 .mu.g/ml. Percent protection provided by
Amplige alone: 4 day, 31%; 5 day, 3%.
TABLE 4 ______________________________________ Inhibition of HIV
Replication by rIFN-.alpha. A and rIFN-.beta. ser 17 with and
without Ampligen cpmRT/ml Avg. cmpRT/ml Effector*
(.times.10.sup.-3) (.times.10.sup.-3) % Decrease
______________________________________ Control-1 877 910 --
Control-2 943 Ampligen-1 896 852 6 Ampligen-2 807 rIFN.alpha.A-1
426 428 53 rIFN.alpha.A-2 430 rIFN.beta.-1 396 398 56 rIFN.beta.-2
400 Ampligen + 41 47 95 rIFN.alpha.A-1 Ampligen + 54 rIFN.alpha.A-2
Ampligen + 41 44 96 rIFN.beta.-1 Ampligen + 48 rIFN.beta.-2
______________________________________ *Equal density cultures of
H9/HTLVIIIB were incubated in duplicate in the presence and absence
of effectors. Conditioned culture fluids were harvested after 48
hours and assayed for reverse transcriptase activity. No inhibition
of cell division was observed. IFNs = 500 .mu./ml Ampligen = 50
.mu.g/ml
The combination of a dsRNA and a lymphokine, administered
separately, including prior administration of the lymphokine to
"prime" the relevant cells, or concurrently, protects cells of the
immune system from viral infection. The degree of protection
conferred by the combination is greater than that conferred by the
dsRNA alone. Importantly, the combination of agents causes no
deletarious inhibition of the normal cell division process.
AMPLIGEN.RTM. is a registered trademark of HEM Research, Inc.,
USA.
* * * * *